Oxidative Damage Control during Decay of Wood by Brown Rot Fungus Using Oxygen Radicals

Abstract

Brown rot wood-degrading fungi deploy reactive oxygen species (ROS) to loosen plant cell walls and enable selective polysaccharide extraction. These ROS, including Fenton-generated hydroxyl radicals (HO˙), react with little specificity and risk damaging hyphae and secreted enzymes. Recently, it was shown that brown rot fungi reduce this risk, in part, by differentially expressing genes involved in HO˙ generation ahead of those coding carbohydrate-active enzymes (CAZYs). However, there are notable exceptions to this pattern, and we hypothesized that brown rot fungi would require additional extracellular mechanisms to limit ROS damage. To assess this, we grew Postia placenta directionally on wood wafers to spatially segregate early from later decay stages. Extracellular HO˙ production (avoidance) and quenching (suppression) capacities among the stages were analyzed, along with the ability of secreted CAZYs to maintain activity postoxidation (tolerance). First, we found that H₂O₂ and Fe²⁺ concentrations in the extracellular environment were conducive to HO˙ production in early (H₂O₂:Fe²⁺ ratio 2:1) but not later (ratio 1:131) stages of decay. Second, we found that ABTS radical cation quenching (antioxidant capacity) was higher in later decay stages, coincident with higher fungal phenolic concentrations. Third, by surveying enzyme activities before/after exposure to Fenton-generated HO˙, we found that CAZYs secreted early, amid HO˙, were more tolerant of oxidative stress than those expressed later and were more tolerant than homologs in the model CAZY producer Trichoderma reesei Collectively, this indicates that P. placenta uses avoidance, suppression, and tolerance mechanisms, extracellularly, to complement intracellular differential expression, enabling this brown rot fungus to use ROS to degrade wood.IMPORTANCE Wood is one of the largest pools of carbon on Earth, and its decomposition is dominated in most systems by fungi. Wood-degrading fungi specialize in extracting sugars bound within lignin, either by removing lignin first (white rot) or by using Fenton-generated reactive oxygen species (ROS) to "loosen" wood cell walls, enabling selective sugar extraction (brown rot). Although white rot lignin-degrading pathways are well characterized, there are many uncertainties in brown rot fungal mechanisms. Our study addressed a key uncertainty in how brown rot fungi deploy ROS without damaging themselves or the enzymes they secrete. In addition to revealing differentially expressed genes to promote ROS generation only in early decay, our study revealed three spatial control mechanisms to avoid/tolerate ROS: (i) constraining Fenton reactant concentrations (H₂O₂, Fe²⁺), (ii) quenching ROS via antioxidants, and (iii) secreting ROS-tolerant enzymes. These results not only offer insight into natural decomposition pathways but also generate targets for biotechnological development.

Keywords: Fenton reaction; antioxidant capacity; glycosyl hydrolases; hydroxyl radicals; oxidative stress tolerance; wood decay.

FIG 1

H₂O₂ and Fe²⁺ contents are staggered as P. placenta decays wood wafers. (A) Schematic of the directional wood colonization system that spatially segregates decay stages. (B) A decrease in wood density and pH at later decay. (C) Contents of H₂O₂ and Fe²⁺ per wood mass (means ± standard deviations; n = 3). Different lowercase letters denote significant differences per two-way randomized ANOVA (α = 0.05). The ratio of H₂O₂ to Fe²⁺ for each wood wafer section is shown at the bottom (higher Fe²⁺ content corresponds to a lower Fenton potential).

FIG 2

Antioxidant capacity increases with progressing brown rot wood decay. (A) The antioxidant capabilities of metabolites in P. placenta were measured as ABTS cation radical scavenging activity, with ascorbic acid (1.0 mg/ml) and trolox (0.12 mg/ml) as positive controls. (B) Potential antioxidant compounds such as phenolics and extracellular polysaccharides (EPS) were evaluated in the fungal extracts (means ± standard deviations; n = 3).

FIG 3

Enzyme oxidative tolerances as relative activities. (A) Damage of GH activities due to the treatment with the Fenton reagents. 0_A, control without Fe²⁺ or H₂O₂; 0_B, control with only 1 mM Fe²⁺ and catalase added from the beginning of the experiment; 0_C, control with only 1 mM FeSO₄ and catalase added after 1 h of incubation; 1, 5, 15, 30, and 60 mM values refer to the concentrations of H₂O₂ used in each treatment, in which the FeSO₄ concentration was fixed at 1 mM. The values are shown as activities of treated samples relative to that of the untreated control (0_A) (means ± standard deviations; n = 3). Bars with the same lowercase letters in the same series are not significantly different (P > 0.05). Initial specific enzyme activities are embedded as units per milligram within the controls. (B) Heat map of oxidative stress tolerance in the brown rot fungus P. placenta relative to that of T. reesei. Relative activities (%) after in vitro Fenton treatment with Fe²⁺ and 1 to 60 mM H₂O₂ are shown as coded colors relative to that of nontreated samples. Mean values of GH activities were used for the heat map analysis.

FIG 4

Treatment controls using only H₂O₂. Damage of GH activities susceptible to the Fenton reaction was assessed for some of the H₂O₂ concentrations used in the Fenton treatment (1, 15, and 60 mM). The values are shown as activities of treated samples (1, 15, and 60 mM H₂O₂) relative to untreated samples (0 mM H₂O₂) (means ± standard deviations; n = 3). Bars with the same lowercase letters in the same series are not significantly different (P > 0.05). Initial specific enzyme activities are embedded as units per milligram within the controls.